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Series GSE80358 Query DataSets for GSE80358
Status Public on Dec 31, 2017
Title PR isoform-specific ER and PR chromatin binding and gene expression observed in-vitro in breast cancer cells.
Organism Homo sapiens
Experiment type Expression profiling by high throughput sequencing
Genome binding/occupancy profiling by high throughput sequencing
Summary Major roadblocks to developing effective progesterone receptor (PR)-targeted therapies in breast cancer include the lack of highly-specific PR modulators, a poor understanding of the pro- or anti-tumorigenic networks for PR isoforms and ligands, and an incomplete understanding of the cross talk between PR and estrogen receptor (ER) signaling. Through genomic analyses of xenografts treated with various clinically-relevant ER and PR-targeting drugs, we describe how the activation or inhibition of PR dictates distinct ER and PR chromatin binding and differentially reprograms estrogen signaling, resulting in the segregation of transcriptomes into separate PR agonist and antagonist-mediated groups. These findings address an ongoing controversy regarding the clinical utility of PR agonists and antagonists, alone or in combination with tamoxifen, for breast cancer management. Genomic analyses of the two PR isoforms, PRA and PRB, indicate that these isoforms bind distinct genomic sites and interact with different sets of co-regulators to differentially modulate gene expression as well as pro- or anti-tumorigenic phenotypes. Of the two isoforms, PRA inhibited gene expression and ER chromatin binding significantly more than PRB. Of note, the two isoforms reprogrammed estrogen activity to be either pro or anti-tumorigenic. In concordance to the in-vitro observations, differential gene expression was observed in PRA and PRB-rich patient tumors and importantly, PRA-rich gene signatures had poorer survival outcomes. In support of antiprogestin responsiveness of PRA-rich tumors, gene signatures associated with PR antagonists, but not PR agonists, predicted better survival outcomes. This differential of better patient survival associated with PR antagonists versus PR agonists treatments was further reflected in the higher anti-tumor activity of combination therapies of tamoxifen with PR antagonists and modulators. Knowledge of various determinants of PR action and their interactions with estrogen signaling to differentially modulate breast cancer biology should serve as a guide to the development of biomarkers for patient selection and translation of PR-targeted therapies to the clinic. 

Overall design For in-vitro experiments, cells were grown in steroid-deprived RPMI for 48 hours to 80% confluence, before being treated for with the hormones of interest (vehicle, 10 nM estrogen, 10 nM R5020 or both estrogen +R5020). Cells were then fixed with 1% formaldehyde for 10 minutes and the crosslinking was quenched with 0.125 M glycine for 5 minutes. Fixed cells were suspended in ChIP lysis buffer (1 ml 1M Tris pH 8.0; 200 µl 5M NaCl; 1 ml 0.5M EDTA; 1 ml NP-40; 1 g SDS, 0.5 g deoxycholate) and sheared in the Diagenode Biorupter for 20 minutes (30 second cycles). 100 µl of sheared chromatin was removed as input control. A 1:10 dilution of sheared chromatin in ChIP dilution buffer (1.7 ml 1M Tris pH 8.0; 3.3 ml 5M NaCl; 5 ml 10% NP-40; 200 µl 10% SDS; to 100 ml with H2O), 4 µg antibody and 30 µl magnetic DynaBeads were incubated in a rotator at 4oC overnight. Chromatin was immunoprecipitated overnight using anti-ER (Santa Cruz Biotechnology HC-20), anti-PR (in-house made KD68) or rabbit IgG (Santa Cruz Biotechnology SC-2027). Next, the immunoprecipitated chromatin was washed with ChIP wash buffer I (2 ml 1M Tris pH 8.0; 3 ml 5M NaCl; 400 µl 0.5M EDTA; 10 ml 10% NP-40; 1 ml 10% SDS; to 100 ml with H2O), ChIP wash buffer II (2 ml 1M Tris pH 8.0; 10 ml 5M NaCl; 400 µl 0.5M EDTA; 10 ml 10% NP-40; 1 ml 10% SDS; to 100 ml with H2O), ChIP wash buffer III (1 ml 1M Tris pH 8.0; 5 ml of 5M LiCl; 200 µl 0.5M EDTA; 10 ml 10% NP-40; 10 ml 10% deoxycholate; to 100 ml with H2O) and TE (pH 8.0). Elution was performed twice from beads by incubating them with 100 µl ChIP-elution buffer (1% SDS, 0.1 M NaHCO3) at 65oC for 15 minutes each. The eluted protein-DNA complexes were de-crosslinked overnight at 65oC in 200 µM NaCl. After de-crosslinking, the mixture was treated with proteinase K for 45 minutes followed by incubation with RNase A for 30 minutes. Finally, DNA fragments were purified using Qiagen PCR purification kit and reconstituted in 50 µl nuclear-free water. Real time PCR was performed using SYBR green. For ChIP-seq library preparations, libraries were prepared using KapaBiosystems LTP library preparation kit (#KK8232) according to the manufacturer’s protocol.
Contributor(s) Greene G, Singhal H
Citation(s) 29435103
Submission date Apr 16, 2016
Last update date May 23, 2019
Contact name Hari Singhal
Organization name University of Chicago
Department Ben May Department for Cancer Research
Lab Geoffrey L Greene
Street address 929 E 57th St
City Chicago
State/province IL
ZIP/Postal code 60637
Country USA
Platforms (1)
GPL16791 Illumina HiSeq 2500 (Homo sapiens)
Samples (50)
GSM2125090 T47D_PRA_Vehicle_RNAseq
GSM2125091 T47D_PRA_E2_RNAseq
GSM2125092 T47D_PRA_R5020_RNAseq
This SubSeries is part of SuperSeries:
GSE80620 Progesterone Receptor Isoforms, Agonists and Antagonists Differentially Reprogram Estrogen Signaling.
BioProject PRJNA318685
SRA SRP073393

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SOFT formatted family file(s) SOFTHelp
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Series Matrix File(s) TXTHelp

Supplementary file Size Download File type/resource
GSE80358_RAW.tar 2.8 Mb (http)(custom) TAR (of BED)
GSE80358_T47D-PRA_RNAseq.csv.gz 124.4 Kb (ftp)(http) CSV
GSE80358_T47D-PRB_RNAseq.csv.gz 62.0 Kb (ftp)(http) CSV
GSE80358_T47D_PRA_reChIP_ERPRAComplexes_R5020_AZ5_subtracted_Veh_and_IgG_.bed.gz 88.1 Kb (ftp)(http) BED
GSE80358_T47D_PRB_reChIP_ERPRBComplexes_R5020_AZ9_subtracted_Veh_and_IgG_.bed.gz 220.6 Kb (ftp)(http) BED
GSE80358_T47D_PR_reChIP_ERPRComplexes_R5020_AZ1_subtracted_Veh_and_IgG_.bed.gz 73.4 Kb (ftp)(http) BED
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Raw data are available in SRA
Processed data provided as supplementary file
Processed data are available on Series record

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